1. Field of the Invention
The present invention relates to a method for synthesis of nucleic acid. In particular, it relates to a method for synthesis of nucleic acid by means of Polymerase Chain Reaction (referred hereinafter as "PCR").
2. Description of the Prior Art
PCR is a method for amplifying a DNA fragment of interest by a factor of hundreds of thousands, in which primers are allowed to bind to a DNA so that a specific region within the DNA is located between the bound primers, then treated with a DNA polymerase, and such a DNA synthesis reaction is repeated. A PCR method is described in Japanese Laid-open Patent Publication No. 61-274697 (1986) of Mullis et al.
A PCR method can be used as a sensitive analysis of nucleic acids in various samples, especially derived from animal body fluids. Thus,a PCR method is used in, for example, diagnosing infections, hereditary diseases or cancers. PCR is also suitable for DNA typing in transplantation or judgement of parenthood. In such cases, peripheral blood is often used as a test material.
A disadvantage of PCR resides in that pigments, proteins, saccharides or unknown impurities inhibit the reaction. Actually, action of many of DNA polymerases, including Taq DNA polymerase, a typical thermostable DNA polymerase derived from Thermus aquaticus, have been well known to be strongly inhibited by a trace of body fluid-derived impurities in a PCR mixture.
Therefore, a PCR method requires an extraction of nucleic acids, before the DNA amplification, from cells, bacteria, viruses or the like (referred generically hereinafter as "gene inclusions") isolated from the subject to be tested. In order to eliminate any inhibitions, the gene inclusions are conventionally lysed by using, for example, enzymes, surfactants or chaotropic agents, and DNAs are then extracted from the lysed gene inclusions by using, for example, phenol or phenol-chloroform.
In recent years, ion exchange resins, glass filters or reagents having a protein-aggregating effect are often used in nucleic acid purification.
It is difficult, however, to remove impurities completely from the samples by these nucleic acid purification procedures. Furthermore, the recovery-quantity of nucleic acids from a sample often varies in these purification procedures. For these reasons, a subsequent nucleic acid synthesis may not sometimes work well, especially when the content of the nucleic acid of interest in the sample is low. In addition, these purification procedures are complicated and time-consuming, and are in danger of contamination.
Thus, a simpler and more effective method for pretreating samples for PCR are required in order to overcome these problems.
Although peripheral blood is often used as a test material for a gene examination, mucous membrane cells of the mouth cavity are superior to peripheral blood as a test material because of their easiness in sampling and the lower risk of infection.
In order to extract DNAs from mucous membrane cells of the mouth cavity, such cells scraped off the mouth cavity with a brush or the like are conventionally treated with a proteolytic enzyme, and DNAs are then purified by means of any one of the above mentioned purification procedures. As mentioned above, however, these procedures have many problems including those in quality and quantity of the DNAs recovered (e.g. an incomplete removal of impurities or a variation in the amount of recovered DNAs) as well as difficulty in treating multiple samples due to the complexity of the procedures, the time-consuming manipulation and the high risk of contamination during the procedures.